During the previous funding cycle, we determined that a UAG-codon, located in the M. barkeri monomethylamine methyltransferase, encoded a new amino acid. Based on its electron density in the native and hydroxylamine complexes, we determined that the amino acid is 4-methyl-(4R,5R)-pyrroline-5- carboxylate, attached to the epsilon nitrogen of lysine. Based on its composition, we named this amino acid, L-pyrrolysine. The subsequent identification, by the Krzycki group, of an amber decoding tRNA(Pyl), and a novel L-pyrrolysyl-tRNA synthetase PylS, that loads L-pyrrolysine onto tRNA(Pyl) make a strong case for Lpyrrolysine being the 22nd genetically-encoded amino acid in nature. During this funding cycle, we propose to continue our efforts in understanding L-pyrrolysine's functional role and its mechanism of incorporation into proteins. This will include: (1) structural studies of L-pyrrolysine containing proteins, (2) structural studies involved in L-pyrrolysine incorporation into protein, and (3) chemical synthesis of L-pyrrolysine and its analogs.